ELISA Kit for serodiagnosis of Q Fever

Q fever affects human beings, cattle, sheep, and goats in particular. The aetiological agent, Coxiella burnetii, is a Gram-negative intracellular bacterium that multiplies in macrophage phagolysosomes. Coxiella burnetii can occur in two antigenic forms, namely, a pathogenic Phase I that is isolated from infected people or animals and an avirulent Phase II that is obtained in ovo or in vitro. The two forms of infection – acute and chronic – have different serological profiles. During the acute phase of the disease, the titres of IgG antibody against Phase II antigens are elevated, whereas during the chronic phase of the disease the titres of IgG antibody against Phase I and Phase II antigens are high. In cows, ewes, and goats Q fever is associated with late-term abortions and reproductive problems such as premature births, dead or weakened foetuses, metritis, and infertility. Nevertheless, the serological responses and isolation of the bacterium in a given species are not necessarily correlated with the disease’s clinical expression. Serotests are appropriate for herd screening, but may be difficult to interpret for an individual subject.

II – PRINCIPLE OF THE TEST

The test uses 96-well microtitration plates sensitised by phase I and phase II antigenic extract from Coxiella burnetii cells. The test blood sera and plasma are diluted in the dilution buffer. The milk samples are used undiluted. Samples are added to the plate which is then incubated and washed. The conjugate, protein G peroxidase-labelled, is added to the wells. The plate is incubated a second time at 21°C +/- 3°C. After the second incubation, the plate is washed again and the chromogen (tetramethylbenzidine) is added. This chromogen has the advantage of being more sensitive than the other peroxidase chromogens and not being carcinogenic. If specific Coxiella burnetii immunoglobulins are present in the test sera or milk the conjugate remains bound to the microwell that contains the bacterial antigen and the enzyme catalyses the transformation of the colourless chromogen into a pigmented compound. The intensity of the resulting blue colour is proportionate to the titre of specific antibody in the sample. The signal read off the negative control microwell is subtracted from that of the positive microwell sensitised by the bacterial antigen.

III – COMPOSITION OF THE KIT

– Microplates Two 96-well microtitration plates (24 strips of 8 wells) sensitised by phase I and phase II antigenic extract from Coxiella burnetii cells. – Washing solution: One 100-ml bottle of 20x concentrated washing solution. The solution crystallises spontaneously when cold. If only part of the solution is to be used, bring the bottle to 21°C +/- 3°C until all crystals have disappeared. Mix the solution well and remove the necessary volume. Dilute the buffer 1:20 with distilled or demineralised water. – Dilution buffer: One 50-ml bottle of 5x colored and concentrated buffer for diluting the blood sera, plasma and the conjugate. The bottle’s content is to be diluted with distilled or demineralised water. If a deposit forms at the bottom of the receptacle filter the solution on Whatman filter paper. – Conjugate: One bottle of Protein G horseradish peroxidase-labelled. – Positive reference: One bottle of positive serum. Store this reagent between +2°C and +8°C. – Negative reference: One bottle of negative serum. Store this reagent between +2°C and +8°C. – Tracer: One bottle of tracer. Reconstitute this reagent with 0.5 ml distilled or demineralised water. Once reconstituted, the reagent is stored at -20°C. Divide this reagent up into several portions before freezing it to avoid repeated freeze/thaw cycles. If these precautions are taken, the reagent can be kept for several months. The tracer is a reference sample that can be used to check the intra-laboratory reproducibility of the kit’s batch. Intra-laboratory reproducibility: Degree of agreement between the results of reiterated tests on the same sample with an identical technical protocol in a given laboratory under variable working conditions. – Single component TMB One 25-ml bottle of the chromogen tetramethylbenzidine (TMB). Store between +2°C and +8°C protected from light. This solution is ready to use. – Stop solution: One 15-ml bottle of the 1 M phosphoric acid stop solution.